GSM7977732: Germinating seed, 26h light, rep1, ATAC-seq; Arabidopsis thaliana; ATAC-seq
Germinating seeds were placed between two microscope slides with a small drop of water which were rotated and squeezed together with gentle force until the embryos were released from within the seed. The mixture of embryos and seed coats were then kept in a small beaker containing 4 mL ice-cold water until all of the sample had been processed (1-2 g starting material). Following this, 4 mL 80% sucrose was added and mixed gently by inversion. The solution was split into two 5 mL microcentrifuge tubes and centrifuged at 3,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The embryo-containing supernatant was filtered through a piece of miracloth (EMD Millipore), washed with ice-cold water, and briefly dried from below using a piece of paper towel to absorb excess water. The embryos were flash frozen in a mortar and ground to a fine powder with a pestle. Approximately 500 mg of frozen ground tissue powder was added to 10.5 mL ice-cold nuclear purification buffer (NPB; 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine) with added cOmplete Mini EDTA-free protease inhibitor cocktail (Merck) in a chilled mortar and ground with a chilled pestle. The homogenate was collected with a 10 mL serological pipette and passed through a 100 µm cell strainer, as well as a 40 µm cell strainer, into a 15 mL Falcon tube (Corning) kept on ice. The tube was centrifuged at 1,200 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The supernatant was discarded and the pellet gently resuspended in 1 mL ice-cold nuclear extraction buffer 2 (NEB2; 0.25 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl2, 1% Triton X-100) with added cOmplete Mini EDTA-free protease inhibitor cocktail (Merck) and transferred to a cooled 1.5 mL microcentrifuge tube. The tube was centrifuged at 12,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The supernatant was removed and the pellet resuspended in 300 µL ice-cold NEB2. This was then pipetted as a layer above 300 µL of nuclear extraction buffer 3 (NEB3; 1.7 M sucrose, 10 mM Tris-HCl pH 8, 2 mM MgCl2, 0.15% Triton X-100) with added cOmplete Mini EDTA-free protease inhibitor cocktail (Merck) in a new 1.5 mL microcentrifuge tube. The tube was centrifuged at 16,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The supernatant was removed, the pellet resuspended in 500 µL ice-cold NPB and transferred to a new 1.5 mL microcentrifuge tube. Nuclei concentration and quality was determined using fluorescence microscopy. Briefly, 1 µL DAPI (0.2 µg/µL) was added to 25 µL nuclei in a 0.5 mL microcentrifuge tube and kept in the dark on ice. A fluorescence microscope was used to count nuclei from 10 µL of the DAPI-stained nuclei inside a Neubauer Improved chamber (Merck). Tagmentation and library preparation was performed following the manufacturers instructions in the Diagenode Tagmentation kit (Diagenode). Approximately 50,000 nuclei were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 1,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The nuclei pellet was gently resuspended in 50 µL tagmentation mastermix (25 µL 2X Tagmentation buffer (Diagenode), 16.5 µL PBS pH 7.4, 5 µL nuclease-free water, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, 2.5 µL loaded Tagmentase (Diagenode)) and incubated at 37 ˚C for 30 min, mixing gently every 5 min. The reaction was terminated by the addition of 250 µL Binding Buffer (Diagenode), mixing by pipetting up and down. The mixture was transferred to a Diagenode MicroCHIP Diapure spin column placed in a collection tube (Diagenode) and centrifuged at 10,000 x g for 30 s in a benchtop centrifuge. The flow-through was discarded and 200 µL Wash Buffer (Diagenode) added to the column. The tube was centrifuged as before, flow-through discarded, and the wash step repeated once more. The column was transferred to a 1.5 mL LoBind tube (Eppendorf) and 12 µL DNA Elution Buffer (Diagenode) added directly to the column matrix. The tube was centrifuged once more as before and the final DNA elution stored at -20 ˚C. For library preparation, 10 µL of sample DNA was mixed with 14 µL of nuclease-free water, 25 µL 2X NEBNext PCR mix (New England Biolabs), and 1 µL of a primer index pair from the 24 UDI For Tagmented Libraries - Set I (Diagenode). A first PCR was performed in a thermocycler using the following conditions: 5 min at 72 ˚C, 30 s at 98 ˚C, 5 cycles of 10 s at 98 ˚C, 30 s at 63 ˚C, and 1 min at 72 ˚C. The number of additional required amplification cycles for each sample was determined before continuing. 5 µL of the PCR reaction mix was combined with 5 µL NEBNext PCR mix (New England Biolabs), 3.75 µL nuclease-free water, 1 µL Evagreen dye (Biotium), and 0.25 µL of the previous primer index pair (Diagenode). A qPCR was performed using the following conditions: 30 s at 98 ˚C, and 20 cycles of 10 s at 98 ˚C, 30 s at 63 ˚C, and 1 min at 72 ˚C. The number of cycles was plotted on the x-axis against the fluorescence readout on the y-axis. The point in the curve at which the reaction reached one-third its maximum fluorescence was found, with the matching cycle number (rounded down to the nearest integer value) used as the final number of additional amplification cycles. This was done by placing the PCR reaction back into the thermocycler, heating to 98 ˚C for 30 s, followed by the same cycling conditions as before for the determined number of additional cycles. DNA purification was performed by the addition of 67.5 µL AMPure XP beads (Beckman Coulter) to the PCR reaction in a 1.5 mL LoBind tube (Eppendorf) and vortexed for 1 s. After incubating at RT for 5 min, the tube was placed in a magnetic rack for another 5 min. The supernatant was removed and replaced with 200 µL 80% ethanol, incubating for 30 s. The ethanol wash step was repeated once. The beads were then air-dried for up to 5 min, after which the tube was removed from the magnetic rack and the beads resuspended in 30 µL DNA Elution Buffer (Diagenode) by tapping. This was incubated at RT for 5 min before being placed in the magnetic rack for another 5 min. 28 µL of the elute was transferred to a new 1.5 mL LoBind tube (Eppendorf) and stored at -20 ˚C. 1 µL was used for quantification and quality control using a Bioanalyzer High Sensitivity DNA chip (Agilent).